CHARACTERIZATION AND MUTATION OF IODOTYROSINE DEIODINASE FROM Haliscomenobacter hydrossis FOR DETOXIFICATION OF IODOPHENOLS
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Iodotyrosine deiodinase (IYD) catalyzes deiodination of mono- and diiodotyrosines (I-Tyr and I2-Tyr) to recycle iodide for proper thyroid function in mammals. IYD is also present in all animals and even some bacteria, although their function in lower organisms is not clear. In this study, a representative set of IYD from eukaryotes and prokaryotes was selected to assess their deiodination of iodophenols for bioremediation. Human IYD, bacterial IYD (hhIYD) and archaeal IYD (pfuIYD) exhibit different substrate binding recognition but their catalytic specificity is surprisingly conserved for iodotyrosines. Greater affinity of hhIYD for 2-iodophenol (2IP) compared to human IYD (~20 fold increased in affinity) indicated that interactions from the phenolate anion of 2IP can compensate for the lack of interactions established by the zwitterion of I-Tyr. Deiodination rates of 2IP by hhIYD are still very slow compared to that of I-Tyr suggesting that affinity is not diagnostic of catalytic efficiency. A crystal structure of hhIYD•I-Tyr shows the formation of an active site lid induced by interactions with the zwitterion of I-Tyr. However, the active site lid is not ordered in a structure of hhIYD•2IP indicating that 2IP cannot trigger closure of the active site lid. Reduction of hhIYD in the alternative presence of a substrate analog F-Tyr and 2IP also indicates differences in the ability of the zwitterionic substrate and 2IP to initiate IYD catalysis. A flavin semiquinone (FMNsq) was detected during the reduction of hhIYD in the presence of F-Tyr but accumulation of this same intermediate was not observed in the presence of 2IP. Crystallographic and redox studies demonstrated that 2IP lacks an ability to initiate formation of the active site lid and cannot stabilize the one-electron chemistry for IYD catalysis, which explain its slow deiodination rates. To enable IYD for bioremediation of iodophenols, three hhIYD mutants were generated in hopes of improving the kcat/Km values for iodophenol turnover. However, these mutants did not provide much increase in the catalytic efficiency for deiodination of iodopehnols as their kcat/Km values are ~3 – 17 fold lower than that of the wild-type hhIYD. However, the studies demonstrated a repulsive interaction between Glu91 and the carboxylate of 4-hydroxy-3-iodobenzoate (2IPCOOH) can significantly increase the Km for deiodination of 2IPCOOH. Removal of this repulsive interaction can significantly decrease the Km as evident by a ~4 fold lower Km for deiodination of 2IPCOOH by an E91R mutant compared to the wild type. This result suggests E91 is a potential mutation site for fine-tuning the Km for deiodination of iodophenols. In addition to hhIYD, two IYD-related enzymes (BluB and 3EO8) were selected to examine their native affinity for 2IP. Neither BluB nor 3EO8 bound 2IP with measurable affinity. Further structural analysis of 3EO8 indicated that its active site was relatively small to accommodate 2IP. 3EO8 mutants were therefore generated to enlarge its active site for 2IP coordination. However, the 3EO8 mutants did not improve the affinity for 2IP. A mutant with the highest affinity with 2IP demonstrated only a 20 % increase in affinity compared to the wild-type 3EO8.